Detection of Superoxide and Peroxynitrite in Model Systems and Mitochondria by the Luminol Analogue L-012

Abstract

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10–100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.

• L-012
• Chemiluminescence
• Superoxide
• Peroxynitrite
• Mitochondria
• NO-donors
• CL, chemiluminescence
• DEA/NO, diethylamine NONOate
• DHE, dihydroethidine
• DMSO, dimethylsulfoxide
• DPI, diphenyleneiodonium chloride
• DTPA, diethylenetriaminepentaacetic acid
• HX, hypoxanthine
• L-012, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt
• MnTMPyP, Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride
• MOPS, 3-morpholinopropanesulfonic acid
• PBS, phosphate buffered saline
• PEG-SOD, polyethylene glycolated superoxide dismutase
• PTIO, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide
• ROS, reactive oxygen species
• SIN-1, 3-morpholino sydnonimine
• SMP, submitochondrial particles
• SNP, sodium nitroprusside
• SPE/NO, spermine NONOate
• Tris, tris(hydroxymethyl)aminomethane
• XO, xanthine oxidase